Voltage-gated ion channels produce electrical signals in the so-called excitable cells including nerve, muscle, and endocrine cells. These channels are, however, also present in non-excitable eukaryotic cells (e.g. lymphocytes) and prokaryotes. This phenomenon raises the fundamental question of whether voltage-gated channels may be regulated by a mechanism other than changing the transmembrane voltage. The answers to this question will point to new strategies for achieving therapeutic interventions via the control of voltage-gated ion channels. Previous work from our lab showed that in a heterologous expression system, certain bacterial sphingomyelinases can activate voltage-gated (Kv) Kv1.3 channels whereas others suppress their activity. In T lymphocytes, the activity of Kv1.3 provides crucial support for signaling events that mediate T cell immune responses. Therefore, I will test the hypothesis that sphingomyelinases can regulate T cell status by modulating Kv1.3 activity, activity known to be essential for T cell activation. I will first test, in T lymphocytes, the hypothesis that enzymatic removal of positively charged choline from the membrane phospholipid sphingomyelin activates Kv1.3 channels whereas removal of the negatively charged phosphate group, along with choline, inhibits the channels (Aim#1; spider, bacterial and human enzymes will be used). Furthermore, I will test the hypothesis that removal of choline from sphingomyelin promotes T cell activation whereas removal of phosphate, along with choline, inhibits T cell activation (Aim#2). These studies will be carried out with a combination of electrophysiology, flow cytometry and molecular biology. The studies outlined above represent initial efforts to test an important novel hypothesis that specific lipases regulate T cell activity via modifying membrane phospholipids that interact with voltage sensors of Kv1.3 channels. Through these studies, we will obtain important information leading to new strategies for treating autoimmune diseases, including multiple sclerosis, and bacterial infection. The outcome of the studies will help establish a new conceptual framework for understanding the regulation of voltage-gated ion channel activity in non-excitable cells, laying the foundation for uncovering a potentially general mechanism underlying the regulation of membrane protein function.